1. Field of the Invention
This invention provides substituted pyrimidine compounds that are useful in treating a patient having cancer. The compounds of this invention are useful as multi-enzyme antifolates selectively targeting the folate receptor (FR). Further, a method of making 5- and 6-substituted cyclopenta[d]pyrimidine for nonclassical and classical antifolates as TS and DHFR inhibitors is provided.
2. Description of the Background Art
Folates are indispensable for cell growth and tissue regeneration. Mammalian cells do not synthesize folates de novo and rely on extracellular folates taken up by three major folate uptake systems: (1) The main transport system at physiologic pH is the reduced folate carrier (RFC or SLC19A1). RFC is a member of the major facilitator superfamily of transporters and functions as anion anti-porter; (2) Folate receptors (FRs) α and β are glycosyl phosphatidylinositol-anchored proteins that transport folates by endocytosis.1,2 Compared to RFC, folate uptake by FRs shows restricted tissue distribution. Several tumor cell lines express FRs (kidney, ovary, thymus and others); (3) The third transport system is a proton-folate symporter that functions optimally at acidic pH-proton-coupled folate transporter (PCFT; SLC46A1).3-5 
Antifolates that target folate-dependent biosynthetic pathways function as anti-proliferative agents. Potent inhibitors of a) dihydrofolate reductase (DHFR) [methotrexate (MTX)], b) thymidylate synthase (TS) [raltitrexed (RTX), GW1843U, pemetrexed (PMX)], c) β-glycinamide ribonucleotide formyl transferase (GARFTase) [lometrexol (LMX), PMX] and d) 5-aminoimidazole-4-carboxamide ribonucleotide formyl transferase (AICARFTase) [PMX] are all antifolate drugs that are used in cancer chemotherapy or have been clinically evaluated.6-8 Figurel shows the chemical structures of known clinically evaluated and available antifolate compounds. All of the clinically available antifolates are transported by RFC, a ubiquitously expressed folate transporter, and hence suffer from dose-limiting toxicity which is a major obstacle in the chemotherapy of cancer.8-9 As such, it is desirable to design targeted antifolates that are selectively taken up by transport systems (other than RFC) which have limited expression and function in normal tissues compared with tumors. Additionally, designing drugs that inhibit multiple enzymes in both purine and pyrimidine pathways can circumvent problems of enzyme and consequent tumor resistance.
FIG. 1 shows the structures of known classical antifolates including Methotrexate (MTX), Pemetrexed (PMX), Raltitrexed (RTX), Lometrexol (LMTX) and Pralatrexate.
The antifolates are a class of antiproliferatives widely recognized for their inhibition of folate metabolism. Major antifolate enzyme targets include thymidylate synthase (TS) and dihydrofolate reductase (DHFR). Inhibition of these enzymes suppresses de novo nucleotide biosynthesis, resulting in an imbalance of purine and pyrimidine precursors and rendering cells incapable of undergoing accurate DNA replication, ultimately resulting in cell death. Clinically relevant TS (e.g., pemetrexed, PMX) and DHFR (e.g., methotrexate (MTX) and pralatrexate) inhibitors (see FIG. 1) continue to play important roles in treating hematologic malignancies and solid tumors.13,14 
Antifolates targeting de novo purine nucleotide biosynthesis were also described and include lometrexol ((6R)5,10-dideazatetrahydrofolate, LMTX) (FIG. 1), (2S)-2-((5-(2-((6R)-2-amino-4-oxo-5,6,7,8-tetrahydro-1H-pyrido[2,3-d]pyrimidin-6-yl)ethyl)thiophene-2-carbonyl)amino)pentanedioic acid (LY309887), and (2S)-2-((5-(2-((6S)-2-amino-4-oxo-1,6,7,8-tetrahydropyrimido[5,4-bate][1,4]thiazin-6-yl)ethyl)thiophene-2-carbonyl)amino)pentanedioic acid (AG2034). All of these drugs inhibit the first folate-dependent step in purine nucleotide biosynthesis, catalyzed by β-glycinamide ribonucleotide (GAR) formyltranferase (GARFTase), and have progressed to clinical trials.16,17 However, their toxicities were dose-limiting, most likely a consequence of their cellular uptake and metabolism to polyglutamates in normal tissues.
The reduced folate carrier (RFC) is one of three mechanisms of (anti)folate uptake into mammalian cells. Cellular requirements for folate cofactors for DNA replication provide a plausible explanation for the high levels of RFC in most tumors. However, demands for folates, and consequently RFC, are also shared by normal tissues such that RFC may not be the optimal mechanism for tumor-selective uptake of cytotoxic folate analogues.15 Other cellular uptake mechanisms, notably, the proton-coupled folate transporter (PCFT) and folate receptors (FRs) α and β, are also expressed in tumors and show more restricted expression in normal tissues.16,17 Furthermore, PCFT is a proton symporter such that in the acidic microenvironment generated by glycolytic tumors, membrane transport and selective tumor targeting by this mechanism is enhanced. For FRα, the apical spatial orientation in normal epithelial tissues is often disrupted in tumors such that its basolateral membrane expression in tumors results in exposure to the circulation. These features provide a compelling rationale for developing folate-based therapeutics that target PCFT and FRα for cancer therapy. Examples of FRα-targeted therapies include a monoclonal antibody, Farletuzumab (Morphotech), a cytotoxic folate conjugate, Vintafolide (EC145; Endocyte), and, ONX0801, a classical antifolate that is selectively transported into cells by FRs over RFC and inhibits de novo thymidylate biosynthesis.15,18 